Project Summary Francisella tularensis is a facultative intracellular pathogen and the causative agent of the zoonotic disease tularemia. Inhalation of as few as 10 bacteria is sufficient to cause a lethal pneumonia in humans. Due to its high morbidity and mortality, as well as ease of dissemination, there are concerns that F. tularensis could be used as a bioterrorism agent. As an intracellular pathogen, F. tularensis must evade cellular innate immune detection; however, the molecular details governing the subversion of the host response during infection are poorly understood. Our research has identified TolC as critical for the virulence of F. tularensis. TolC is the outer membrane-spanning portion of both bacterial drug efflux pumps and the type I secretion system, which secretes a wide range of bacterial toxins. Together, our data demonstrate that F. tularensis delays programmed host cell death responses during infection in a TolC-dependent manner, providing extended time for bacterial replication within the protected intracellular niche. Our data suggest that F. tularensis secretes effector molecules via TolC to execute these host suppressive functions. This proposal will characterize the TolC-dependent modulation of programmed host cell death by F. tularensis. The first aim of this proposal will define the contribution of TolC to the virulence of the F. tularensis Schu S4 strain, a human pathogen. The second aim of the proposal will analyze the cellular signaling pathways that are subverted during F. tularensis infection. The third aim will identify virulence factors secreted via TolC that are important for innate immune evasion. Data generated by this proposal will lead to a detailed, mechanistic understanding of TolC function in F. tularensis. The identification of toxins secreted by F. tularensis would represent a significant advance for the field. In addition, the proposed research will provide new insights into strategies used by intracellular pathogens to manipulate host responses.